RNA REGULATORY DOMAINS: FOLDING, RECOGNITION & CATALYSIS

Sloan-Kettering Inst Can Research, New York NY

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Abstract

DESCRIPTION (provided by applicant): This revised application focuses on 3 projects addressing structural and functional aspects of folding, recognition and catalysis by RNA regulatory domains. Project 1 focuses on the structural characterization of the core domains of metabolite-sensing mRNAs discovered in the Ronald Breaker laboratory, which adopt complex junctional topologies capable of ligand-induced functional modulation. Our structural research will initially focus on the free and bound core domains of guanine/adenine-sensing and thiamine pyrophosphate-sensing mRNAs, for which we have collected promising NMR and crystallographic data. Our structural studies combined with functional efforts in the Breaker laboratory should highlight the principles of molecular recognition and metabolite encapsulation by mRNA, and define the allosteric mRNA transitions associated with the modulation of gene expression levels and metabolic homeostasis. Project 2 on ribozymes catalyzing chemical reactions, focuses on the structural characterization of RNA motifs with Diels-Alderase catalytic activities discovered in the Andres Jaschke laboratory. We are attempting to structurally characterize the catalytic RNA scaffold in the context of bound substrates, transition state analogs and products, with promising crystallographic and NMR data collected on the product complex. Our structural characterization of the Diels-Alderase ribozyme, together with mutational and energetics studies in the Jaschke laboratory, should identify principles for generation of novel ribozymes with controllable catalytic activities, tunable specificites and enantiomeric capabilities. Project 3 focuses on protein-RNA recognition events that mediate the degradation of metazoan histone mRNA, a process tightly coupled to cell cycle progression. Histone mRNAs contain a unique bipartite stem-loop scaffold followed by an ACCA sequence, whose stem, loop and flanking sequences are targeted along opposite faces, both separately and simutaenously, by a stem-loop binding protein and a histone mRNA 3' end-specific exonuclease. Our proposed structural and energetics characterization of binary and ternary protein-RNA recognition events involved in histone mRNA 3'-end recognition and cleavage should provide insights into the mechanisms that cells use to achieve precise cell cycle-regulated mRNA degradation.